WO2015103479A1 - Compositions antimicrobiennes comprenant des anticorps monodomaines et l'exotoxine de pseudomonas - Google Patents

Compositions antimicrobiennes comprenant des anticorps monodomaines et l'exotoxine de pseudomonas Download PDF

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WO2015103479A1
WO2015103479A1 PCT/US2015/010045 US2015010045W WO2015103479A1 WO 2015103479 A1 WO2015103479 A1 WO 2015103479A1 US 2015010045 W US2015010045 W US 2015010045W WO 2015103479 A1 WO2015103479 A1 WO 2015103479A1
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vhh
fragment
heavy chain
chain immunoglobulin
cells
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PCT/US2015/010045
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Richard Markham
Eileen GEOGHEGAN
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The Johns Hopkins University
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Priority to US15/108,380 priority Critical patent/US10010625B2/en
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Priority to US16/007,052 priority patent/US10376596B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6839—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting material from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6813—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin the drug being a peptidic cytokine, e.g. an interleukin or interferon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A61K47/6829—Bacterial toxins, e.g. diphteria toxins or Pseudomonas exotoxin A
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
    • C07K16/085—Herpetoviridae, e.g. pseudorabies virus, Epstein-Barr virus
    • C07K16/087—Herpes simplex virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin

Definitions

  • variable domain of heavy-chain only antibodies found in members of the camelid family represents the smallest naturally occurring functional domain of the antibody molecule.
  • VHH variable domains
  • VHH have the same antigen binding capability as full-length antibodies, yet are typically around 15 kDa in size.
  • VHH demonstrate remarkable stability under a wide range of denaturing, temperature, and pH conditions.
  • VHHs exhibit increased solubility compared to full-length antibodies or other antibody fragments, and very high expression levels have been achieved in E. coli, yeast, and tobacco expression systems. Due to a high degree of sequence homology between camelid and other mammalian variable domains, VHH have been shown not to be immunogenic in mice.
  • VHH have enhanced tissue penetration, and an extended CDR3 loop allows VHH access to cryptic epitopes in enzymatically active sites that are unavailable for binding by full length antibodies.
  • VHH have been promoted as promising biomedical tools.
  • a myriad of VHH have been successfully developed for diverse purposes including diagnostics, imaging, and biochemical and therapeutic applications.
  • VHH directed against viruses, bacteria, protozoa, and fungi have all been identified.
  • VHH can act as a monomeric domain, or they can be expressed in a multivalent context to increase avidity and activity.
  • bispecific VHH can be assembled that bind different epitopes, which can in some cases dramatically increasing neutralization efficacy.
  • HSV-2 is one of the most prevalent sexually transmitted infections (STIs) in the world, and recent estimates indicate that roughly 16% of people ages 15-49 worldwide are infected. There has been great interest in the development of a prophylactic vaccine to prevent HSV-2 infection over the past several decades, but unfortunately, an effective one has yet to be developed.
  • STIs sexually transmitted infections
  • HIV-1 Human immunodeficiency virus type I
  • bnAbs broadly neutralizing antibodies
  • Env to CD4 initiates a series of conformational changes of the Env structure, leading to exposure and/or formation of coreceptor binding sites that are recognized by cell surface co-receptors (e.g. chemokine receptors CCR5 or CXCR4).
  • cell surface co-receptors e.g. chemokine receptors CCR5 or CXCR4.
  • a microbicide is a substance that can be applied to mucosal surfaces, including the vagina and rectum, to prevent infection with an STL
  • a significant public health goal has been to try develop a successful microbicide against HSV-2 and HIV-1, including vaginal delivery of antiviral drugs, antibody-based strategies, and small-interfering R As. It has been demonstrated that vaginally applied monoclonal antibodies and single chain antibody variable fragments (scFv) directed against gD2 protect against HSV-2 infection in animal models. The issue of how to vaginally deliver a neutralizing antibody against HSV2 or HIV-1 without the direct application of the antibody immediately prior to sexual intercourse has yet to be resolved, however. Furthermore, the current methods of production of monoclonal antibodies and scFvs can be cost-prohibitive to scale up, as antibodies are complex molecules with multiple protein chains that are not easily purified and assembled.
  • VHH antibodies are small enough to be secreted by many types of commensal bacteria, including, for example, Lactobacilli, an ideal organism for delivery of VHH antibodies because they are a major component of the vaginal flora and because systems for expression of heterologous proteins have been developed for these bacteria.
  • the inventors Using a VHH antibody that bound to gD2 of HSV-2, in an embodiment, the inventors created a P. aeruginosa Exotoxin A (PE)-based immunotoxin that specifically targets HSV-2 infected cells. This immunotoxin specifically binds to cells expressing gD2 at the cell surface, causing internalization of the entire protein, allowing the exotoxin A portion to act by halting protein synthesis, ultimately resulting in cell death.
  • PE P. aeruginosa Exotoxin A
  • the present invention provides a heavy chain immunoglobulin of the VHH type or fragment thereof having an amino acid sequence of at least 85% identity to SEQ ID NO: 3 (R33) and having affinity for glycoprotein D2 (gD2) of HSV-2 or antigen thereof.
  • the present invention provides a heavy chain immunoglobulin of the VHH type or fragment thereof having an amino acid sequence of at least 85% identity to SEQ ID NO: 4 (R33) and having affinity for glycoprotein D2 (gD2) of HSV-2 or antigen thereof that is covalently linked to the P. aeruginosa Exotoxin A subunit or a functional portion or fragment thereof.
  • the present invention provides a multimeric molecule comprising a heavy chain immunoglobulin fragment of the VHH type as described herein, in which VHH sequences are fused to yield multimeric units of 2 or more VHH units optionally linked via a spacer molecule.
  • the present invention provides a multimeric molecule comprising two or more VHH sequences as described herein, which are fused to yield 2, 3, 4 or 5 or more VHH units optionally linked via a spacer molecule.
  • the present invention provides an expression vector comprising the gene encoding the heavy chain immunoglobulin fragment described herein.
  • the present invention provides a heavy chain immunoglobulin of the VHH type or fragment thereof comprising an amino acid sequence of at least 85% identity to SEQ ID NOS. 7 or 1 l,and having affinity for envelope proteins of HIV- 1.
  • the present invention provides a nucleic acid encoding a heavy chain immunoglobulin fragment of the VHH type and having affinity for envelope proteins of HIV- 1 which is covalently linked to the P. aeruginosa Exotoxin A subunit comprising the nucleic acid sequence of SEQ ID NO: 6.
  • the present invention provides a method for the therapy or prophylaxis of HIV- 1 infection, comprising administering to a patient a heavy chain immunoglobulin or fragment thereof having affinity for envelope proteins of HIV- 1 which is covalently linked to the P. aeruginosa Exotoxin A subunit or functional portion or fragment thereof, or the multimeric molecule of described herein.
  • the present invention provides a method for the therapy or prophylaxis of HIV- 1 infection, comprising administering to a patient a micro-organism expressing the heavy chain immunoglobulin or fragment thereof and having affinity for envelope proteins of HIV- 1 or an antigen thereof that is covalently linked to the P. aeruginosa Exotoxin A subunit or functional portion or fragment thereof, or the multimeric molecule described herein.
  • Figure 3 shows antibody reactivity to purified gD2 (ELISA).
  • ELISA wells were coated with gD2 and detected with a panel of anti-gD2 antibodies: R45 (rabbit, polyclonal), HSV8 (human, monoclonal), DL6 (mouse, monoclonal), anti-His (mouse, monoclonal). Additionally wells coated with gD2 where only HRP-conjugated secondary antibody (anti- rabbit, anti-human, and anti-mouse) was added were run as controls.
  • Figure 4 depicts llama serum ELISA.
  • Llama serum collected before the initiation of immunization (naive) and after each immunization (Im# 1-5) was diluted 1 : 10,000 and used to coat ELISA wells.
  • gD2 was added and binding was detected to determine if the llamas mounted an immune response against the gD2 immunizations.
  • Figure 8 is a flowchart diagramming the process of identifying unique VHH sequences that bind to gD2. Individual phage clones were amplified and tested by ELISA to determine if they are reactive to gD2. Those that were reactive were sequenced, and the sequences were compared to determine the number of unique VHH sequences.
  • Figure 10 illustrates antibody capture biopanning.
  • FIG. 1 1 shows the results of antibody capture biopanning VHH-Phage ELISA.
  • VHH-phage clones after three rounds of capture biopanning were individually amplified and tested for reactivity to gD2 by ELISA.
  • Wells were coated with gD2 and VHH-phage clones were added and then detected with an anti-phage antibody.
  • Previously identified VHH-phage were used as positive (R33 and P4) and negative (P 10) controls.
  • the anti-gD2 antibody DL6 was also used as a positive control.
  • Each VHH-phage was assayed in duplicate and error bars represent maximum and minimum values.
  • FIG. 12 illustrates a unique VHH amino acid sequence alignment.
  • VHH inserts originally amplified from variable region of heavy chain only antibodies, were sequenced from VHH-phage clones and aligned to determine unique VHH sequences identified from the gD2 biopanning process.
  • FIG. 13 depicts expression and purification of VHH from E. coli.
  • E. coli were transformed with VHH/pET plasmids and small scale cultures were grown and induced to determine solubility of VHH proteins.
  • a representative gel demonstrating that VHH derived from one llama are located in the pellet (P), while VHH derived from a second llama are located in both the supernatant (SN) and the pellet.
  • a representative gel demonstrated the size and purity of purified R33 and bvR33.
  • Figure 14 shows that purified VHH bind to gD2.
  • ELISAs were performed in which wells were coated with VHH and gD2 was added to assay for their ability to bind gD2. Each dilution was assayed in duplicate and error bars represent maximum and minimum values.
  • Figure 15 depicts VHH binding to gD2-expressing cell line.
  • z4/6 cells surface expression of gD2
  • VHH surface expression of gD2
  • C R33, D: P4, E: bvR33, F: R15
  • DL6 was used as a positive control to verify that gD2 was expressed (A)
  • B secondary antibody control with no VHH or primary antibody was also used as a negative control
  • Figure 16 depicts the purification of pentavalent VHH.
  • NR33 verotoxin B subunit
  • Figure 17 shows the VHH neutralization of HSV-2 using the present invention.
  • Virus was incubated with dilutions of VHH for 1 hour at 37 °C and then plated on Vero cells to assay for VHH neutralizing activity. Each dilution was assayed in duplicate and error bars represent maximum and minimum plaque numbers. Results are expressed as percent inhibition compared to plaque numbers from untreated virus. Statistical significance compared to untreated virus was calculated by ANOVA and is indicated by asterisks (P ⁇ 0.05). The known neutralizing antibody HSV8 was used in graph A as a positive control.
  • FIG. 18 shows VHHExoA were purified from the insoluble fraction of induced E. coli cells and refolded according to previously published protocols.
  • FIG. 19 illustrates that VHH and VHHExoA Bind to gD2.
  • a capture ELISA was performed to determine if the VHH portion of R33ExoA is able to bind gD2 when expressed with a C-terminal exotoxin A. Each dilution was assayed in duplicate and error bars represent maximum and minimum values.
  • Figure 20 shows the toxicity of VHHExoA on Vero cells and z4/6 cells.
  • 20A Dilutions of VHH-ExoA proteins were added to Vero cells (do not express gD2) and their cytotoxicity was measured by addition of MTS reagent (Promega, Madison, WI). Triton X- 100 was added at 0.05% to the first dilution to serve as a positive control for cytotoxicity, and it diluted as the other samples were. Dilutions of each protein were added to wells in triplicate and error bars represent standard deviation.
  • VHH-ExoA proteins were added to z4/6 cells (express gD2) and their cytotoxicity was measured by addition of MTS reagent (Promega, Madison, WI). Triton X-100 was added at 0.05% to the first dilution to serve as a positive control for cytotoxicity, and it diluted as the other samples were.
  • Figure 21 provides results of a VHHExoA infectious center assay.
  • HSV-2 infected Vero cells were treated with dilutions of VHHExoA, R33, or PBS for about 16 hours. Infected cells were then harvested and diluted in uninfected Vero cells to assay for the number of infectious centers that remain. This is a representative graph from four independent experiments. Error bars represent standard error of the mean.
  • Figure 22 depicts a nucleic acid sequence (22A) and an amino acid sequence (22B) for an embodiment of the VHHR33ExoA construct of the present invention.
  • Figure 23 depicts a nucleic acid sequence (23A) and an amino acid sequence (23B) for an embodiment of the J3VHHExoA construct of the present invention.
  • Figure 24 depicts the amino acid sequence of an embodiment of the present invention comprising fully expressed J3VHH construct (SEQ ID NO: 11) and the
  • J3VHHExoA construct (SEQ ID NO: 12).
  • the figures show the annotated protein sequence of insert containing J3 VHH fused to Exotoxin A Key: Gray highlight signifies the start codon; Purple Highlight is the His Tag; Highlighted yellow regions contain restriction sites; 5' end EcoRl ; 3' end Avrll; Underlined sequence is the J3 VHH; Italicized sequence is the linker region; Includes yellow highlighted multiple cloning site sequence; Includes myc tag highlighted in blue; Bold sequence is the P. aeruginosa Exotoxin A subunit; Red highlight is the stop codon.
  • Figure 25 depicts cell viability graphs showing the viability of CHO cells expressing or not expressing envelope after exposure to either the J3 VHH or J3 VHH fused to exotoxin A.
  • Figure 26 shows the relative neutralizing capability of J3 and J3ExoA. HIV-1 AD8 virus were produced from 293T cells and incubated with indicated concentration of J3 or J3ExoA for 1 h, and then infected MAGI-CCR-5 cells. Viral infection was determined by MAGI assay. The infectivity of AD8 in absence of any protein was set as 100%.
  • Figure 27 shows Relative ability of 50 nM J3 and J3ExoA to reduce viral load in PBMC culture when exposed to cells pre-infected with HIVBaL.
  • the present invention provides a heavy chain immunoglobulin of the VHH type or fragment thereof having an amino acid sequence of at least 85% identity to SEQ ID NO. 3 (R33) and having affinity for glycoprotein D 2 (gD2) of HSV-2 or antigen thereof.
  • variable domain of the VHH type or fragment thereof means the variable domain of heavy-chain only antibodies found in members of the camelid family, and which represents the smallest naturally occurring functional domain of the antibody molecule.
  • VHH variable domains
  • These variable domains, termed VHH have the same antigen binding capability as full-length antibodies, yet are typically around 15 kDa in size.
  • VHH have enhanced tissue penetration, and an extended CDR3 loop allows VHH access to cryptic epitopes in enzymatically active sites that are unavailable for binding by full length antibodies.
  • VHH can also serve as carriers for other molecules through conjugation or expression as a fusion protein with an effector domain to create an immunoconjugate.
  • immunoconjugate is a conjugate of a binding molecule (e.g., an antibody) with an atom, molecule, or a higher-ordered structure (e.g., with a liposome), and an antigen, and/or therapeutic agent, and/or a diagnostic agent.
  • a binding molecule e.g., an antibody
  • an atom, molecule, or a higher-ordered structure e.g., with a liposome
  • the present invention provides a heavy chain immunoglobulin of the VHH type or fragment thereof having an amino acid sequence of at least 85% identity to SEQ ID NO. 4 (R33) and having affinity for glycoprotein D 2 (gD2) of HSV-2 or antigen thereof that is covalently linked to the P. aeruginosa Exotoxin A subunit.
  • polypeptide as used herein includes oligopeptides and refers to a single chain of amino acids connected by one or more peptide bonds.
  • a peptide or polypeptide fragment thereof, capable of being cleaved by a specific protease means an amino acid sequence which is specifically recognized by a protease enzyme and specifically binds and hydrolytically cleaves that amino acid sequence.
  • the peptide sequence can be any sequence of between about 3 to about 20 amino acids in length, which is known to be cleaved by a known protease.
  • the present invention provides an immunoconjugate where the peptide or polypeptide fragment thereof, capable of being cleaved by a specific protease is an amino acid sequence cleaved by a protease normally found on cancer cell membranes.
  • the protease is furin, which is found on many types of tumor cells.
  • the term "functional portion" when used in reference to a monoclonal antibody or antigenic epitope refers to any part or fragment, which part or fragment retains the biological activity of which it is a part (the parent molecule, antibody, or antigen).
  • Functional portions encompass, for example, those parts that retain the ability to specifically bind to the antigen (e.g., in an MHC-independent manner), or detect, treat, or prevent the disease, to a similar extent, the same extent, or to a higher extent, as the parent molecule.
  • the functional portion can comprise, for instance, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more, of the parent molecule.
  • the functional portion can comprise additional amino acids at the amino or carboxy terminus of the portion, or at both termini, which additional amino acids are not found in the amino acid sequence of the parent molecule.
  • the additional amino acids do not interfere with the biological function of the functional portion, e.g., specifically binding to a cancer antigen, having the ability to detect cancer, treat or prevent cancer, etc. More desirably, the additional amino acids enhance the biological activity, as compared to the biological activity of the parent molecule.
  • protein is meant a molecule comprising one or more polypeptide chains.
  • the invention also provides an immunoconjugate molecule comprising at least one of the polypeptides described herein along with at least one other polypeptide.
  • the other polypeptide can exist as a separate polypeptide of the fusion protein, or can exist as a polypeptide, which is expressed in frame (in tandem) with one of the inventive polypeptides described herein.
  • the other polypeptide can encode any peptidic or proteinaceous molecule, or a portion thereof. Suitable methods of making fusion proteins are known in the art, and include, for example, recombinant methods. See, for instance, Choi et al., o/. Biotechnol. 31 : 193-202 (2005).
  • recombinant antibody refers to a recombinant (e.g., genetically engineered) protein comprising at least one of the polypeptides of the invention and a polypeptide chain of an antibody, or a portion thereof.
  • the polypeptide of an antibody, or portion thereof and is a heavy chain immunoglobulin of the VHH type or fragment thereof.
  • the polypeptide chain of an antibody, or portion thereof can exist as a separate polypeptide of the recombinant antibody.
  • the polypeptide chain of an antibody, or portion thereof can exist as a polypeptide, which is expressed in frame (in tandem) with the polypeptide of the invention.
  • the polypeptide of an antibody, or portion thereof can be a polypeptide of any antibody or any antibody fragment, including any of the antibodies and antibody fragments described herein.
  • the present invention provides a heavy chain immunoglobulin of the VHH type or fragment thereof comprising an amino acid sequence of at least 85% identity to SEQ ID NOS. 8 or 12, and having affinity for envelope proteins of HIV- 1 which is covalently linked to the P. aeruginosa Exotoxin A subunit or functional portion or fragment thereof.
  • the term "functional variant” as used herein refers to an immunoconjugate, polypeptide, or protein having substantial or significant sequence identity or similarity to a parent immunoconjugate, polypeptide, or protein, which functional variant retains the biological activity of the immunoconjugate, polypeptide, or protein of which it is a variant.
  • the functional variant can, for instance, be at least about 30%, 50%, 75%, 80%, 90%, 98% or more identical in amino acid sequence to the parent immunoconjugate, polypeptide, or protein.
  • the functional variants can comprise the amino acid sequence of the parent immunoconjugate, polypeptide, or protein with at least one non- conservative amino acid substitution.
  • the non-conservative amino acid substitution it is preferable for the non-conservative amino acid substitution to not interfere with or inhibit the biological activity of the functional variant.
  • the non-conservative amino acid substitution enhances the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the parent immunoconjugate, polypeptide, or protein.
  • inventive immunoconjugates, polypeptides, and proteins can be synthetic, recombinant, isolated, and/or purified.
  • the present invention provides a nucleic acid molecule which encodes the immunoconjugates described above.
  • the present invention includes nucleic acid molecules comprising SEQ ID OS: 1, 2, 5 and 6.
  • nucleic acid comprising a nucleotide sequence encoding any of the immunoconjugates, polypeptides, or proteins described herein (including functional portions and functional variants thereof).
  • the nucleic acid does not comprise any insertions, deletions, inversions, and/or substitutions. However, it may be suitable in some instances, as discussed herein, for the nucleic acid to comprise one or more insertions, deletions, inversions, and/or substitutions.
  • the nucleic acids of the invention are recombinant.
  • the term “recombinant” refers to (i) molecules that are constructed outside living cells by joining natural or synthetic nucleic acid segments to nucleic acid molecules that can replicate in a living cell, or (ii) molecules that result from the replication of those described in (i) above.
  • the replication can be in vitro replication or in vivo replication.
  • the nucleic acids can be constructed based on chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. See, for example, Sambrook et al., supra, and Ausubel et al, supra.
  • a nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides).
  • modified nucleotides that can be used to generate the nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl) uracil, 5- carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil,
  • dihydrouracil beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1 -methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5- methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'- methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil- 5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2- thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic
  • the substituted nucleic acid sequence may be optimized. Without being bound to a particular theory, it is believed that optimization of the nucleic acid sequence increases the translation efficiency of the mR A transcripts. Optimization of the nucleic acid sequence may involve substituting a native codon for another codon that encodes the same amino acid, but can be translated by tR A that is more readily available within a cell, thus increasing translation efficiency. Optimization of the nucleic acid sequence may also reduce secondary mRNA structures that would interfere with translation, thus increasing translation efficiency.
  • the invention also provides an isolated or purified nucleic acid comprising a nucleotide sequence which is complementary to the nucleotide sequence of any of the nucleic acids described herein or a nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence of any of the nucleic acids described herein.
  • nucleic acids of the invention can be incorporated into a recombinant expression vector.
  • the invention provides recombinant expression vectors comprising any of the nucleic acids of the invention.
  • “recombinant expression vector” means a genetically-modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA, protein, polypeptide, or peptide by a host cell, when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed within the cell.
  • the vectors of the invention are not naturally-occurring as a whole. However, parts of the vectors can be naturally-occurring.
  • the inventive recombinant expression vectors can comprise any type of nucleotides, including, but not limited to DNA and RNA, which can be single-stranded or double-stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides.
  • the recombinant expression vectors can comprise naturally-occurring, non-naturally-occurring internucleotide linkages, or both types of linkages.
  • the non-naturally occurring or altered nucleotides or internucleotide linkages do not hinder the transcription or replication of the vector.
  • the recombinant expression vector can comprise a native or nonnative promoter operably linked to the nucleotide sequence encoding the immunoconjugate, polypeptide, or protein (including functional portions and functional variants thereof), or to the nucleotide sequence which is complementary to or which hybridizes to the nucleotide sequence encoding the immunoconjugate, polypeptide, or protein.
  • promoters e.g., strong, weak, inducible, tissue-specific and developmental-specific, is within the ordinary skill of the artisan.
  • the combining of a nucleotide sequence with a promoter is also within the skill of the artisan.
  • the promoter can be a non-viral promoter or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, and a promoter found in the long-terminal repeat of the murine stem cell virus.
  • CMV cytomegalovirus
  • VHH linked immunotoxin can be used in multiple ways. If applied vaginally, an anti-gD2 immunotoxin could prevent HSV-2 infection by killing infected epithelial cells prior to establishment of latency.
  • the immunotoxins of the present invention have the potential to not only act as a microbicides to prevent initial infection, but can also act to reduce viral shedding in infected individuals by eliminating gD2-expressing cells during reactivation of the virus from latency.
  • the present invention provides a method for treating HSV2 in a subject, comprising administering to the subject, a therapeutically effective amount of the immunoconjugate described above and a pharmaceutically acceptable carrier.
  • the present invention provides a method for treating HIV-1 in a subject, comprising administering to the subject, a therapeutically effective amount of the immunoconjugate described above and a pharmaceutically acceptable carrier.
  • the binding affinity of the VHH will be enhanced by converting it from a monovalent to a bivalent VHH (bvJ3). This will be done, by using appropriate primer sets to amplify a second J3 sequence and incorporate a GS linker between the two J3 sequences. DNA encoding the 38 kd fragment of ExoA will then be cloned in frame to the C terminus of the VHH. Using dilution series, we will then test the relative killing activity of the bvJ3-ExoA and J3-ExoA using the Env+ and Env- CHO cell lines. The expectation is that the bvJ3-ExoA will be active at lower concentrations. A similar construct will be developed using the active fragment of diphtheria toxin.
  • albumin-binding construct If bioactivity of the albumin-binding construct is confirmed, its albumin binding will be evaluated by ELISA, testing the binding of the ABP-VHH-ExoA construct in wells coated with albumin vs. control wells, as described, using antibodies targeting the His-tag incorporated into the VHH construct for ELISA development.
  • the exotoxin can be administered subcutaneous ly by being incorporated into sustained delivery particles.
  • the Medusa® drug delivery platform consists of proprietary depot hydrogels for the formulation and/or the extended release of a broad range of biologies (including proteins, antibodies, peptides and vaccines) and of small molecules (injectable drugs). These hydrogels have been proven to be safe and biodegradable. Medusa enables the controlled delivery from 1 day up to 14 days of non-denatured or non-modified drugs that maintain full bioactivity. The in vivo efficacy of the embodiments can be confirmed by Western blotting of serum obtained from mice at different time points post administration of either the "native" J3-ExoA or J3-ExoA administered in an extended release format.
  • the immunoconjugates of the present invention can be formulated into a composition, such as a pharmaceutical composition.
  • the invention provides a pharmaceutical composition comprising any of the immunoconjugates, polypeptides, proteins, functional portions, functional variants, nucleic acids, expression vectors, and a pharmaceutically acceptable carrier.
  • the inventive pharmaceutical compositions containing any of the inventive immunoconjugates can comprise more than one immunoconjugate.
  • the carrier is a pharmaceutically acceptable carrier.
  • the carrier can be any of those conventionally used and is limited only by chemico-physical considerations, such as solubility and lack of reactivity with the active compound(s), and by the route of administration.
  • the pharmaceutically acceptable carriers described herein, for example, vehicles, adjuvants, excipients, and diluents, are well-known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the active agent(s) and one which has no detrimental side effects or toxicity under the conditions of use.
  • immunoconjugate Accordingly, there are a variety of suitable formulations of the pharmaceutical composition of the invention.
  • suitable formulations for aerosol, parenteral, subcutaneous, interperitoneal, vaginal and rectal, administration are exemplary and are in no way limiting. More than one route can be used to administer the
  • a particular route can provide a more immediate and more effective response than another route.
  • the preferred route is vaginal.
  • the immunoconjugate of the invention can be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or liposomes.
  • the amount or dose of the immunoconjugate administered should be sufficient to effect, e.g., a therapeutic or prophylactic response, in the subject or animal over a reasonable time frame.
  • the dose of the immunoconjugate administered should be sufficient to effect, e.g., a therapeutic or prophylactic response, in the subject or animal over a reasonable time frame.
  • immunoconjugate should be sufficient to bind to a target antigen, or detect, treat or prevent an infection in a period of from about 2 hours or longer, e.g., 12 to 24 or more hours, from the time of administration. In certain embodiments, the time period could be even longer.
  • the dose will be determined by the efficacy of the particular immunoconjugate and the condition of the animal (e.g., human), as well as the body weight of the animal (e.g., human) to be treated.
  • multiple administrations of the immunoconjugate can be required to effect elimination of the viral burden in the subject. For example, there may be an initial dose followed by a period of time where the viral or tumor burden is monitored and then subsequent dosages of the immunoconjugate are given in an iterative fashion.
  • inventive methods can provide any amount of any level of treatment or prevention of cancer in a mammal.
  • the treatment or prevention provided by the inventive method can include treatment or prevention of one or more conditions or symptoms of the disease, e.g., cancer, being treated or prevented.
  • prevention can encompass delaying the onset of the disease, or a symptom or condition thereof.
  • the term "subject” refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). It is more preferred that the mammals are from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). It is most preferred that the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). An especially preferred mammal is the human.
  • mammals of the order Rodentia such as mice and hamsters
  • mammals of the order Logomorpha such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). It is
  • NUNC Maxisorp ELISA plates (Thermo Fisher Scientific Inc., Waltham, MA) were coated with 100 ⁇ of gD2 at 10 ⁇ g/mL and incubated ON at 4 °C. The plate was blocked with 2% BSA in PBS for 30 minutes at RT. Freshly thawed serum samples were diluted 1 : 10,000 in PBS and added in duplicate to wells for 1 hour at RT. Wells were washed 5 x 200 ⁇ PBS-T per well and HRP-conjugated anti-llama secondary antibody (Bethyl Laboratories, Inc) was diluted 1 : 10,000 in PBS-T and added to wells for 1 hour at RT.
  • HRP-conjugated anti-llama secondary antibody Bethyl Laboratories, Inc
  • Vero cells were plated in Falcon 12-well trays (Thermo Fisher Scientific Inc., Waltham, MA) at 4xl0 6 cells per tray and incubated ON at 37 °C. Llama serum samples were heat inactivated at 56 °C for 60 minutes and serial two-fold dilutions were made in DMEM/2% FBS. Approximately 5000 pfu/mL of HSV-2 G was added to each dilution and all dilutions were incubated at 37 °C for 1 hour. Media was removed from the Vero cells and the serum dilutions with virus were added in duplicate to cells for 1 hour at 37 °C, with gentle shaking every ten minutes to distribute volume over cells.
  • Nested PCR was performed to amplify the VHH regions from the genomic DNA using primers that bind to the conserved regions flanking the VHH genes. The first round of PCR was performed with primers as previously published, while the second round of primers introduced the appropriate restriction sites for ligation into the phage genome.
  • Anti-T7 tail fiber monoclonal antibody (GE Healthcare Life Sciences, Piscataway, NJ) was diluted to 1 : 1000 and added to each well for 1 hour at RT. After washing the plate 5 x 200 PBS-T per well, HRP -conjugated anti-mouse IgG secondary antibody (Jackson ImmunoResearch, West Grove, PA) was added at 1 :3000 and incubated at RT for 1 hour. After a final wash of 5 x 200 PBS-T per well, 200 ⁇ , of ABTS® ELISA HRP Substrate (KPL, Gaithersburg, MD) was added. The plate was read at 405 nm using a BioTek Synergy HT Plate Reader (Winooski, VT).
  • VHH sequences were amplified from phage by PCR amplification using the primers that introduced EcoRI and Xhol restriction sites for cloning in to pET-47b (Novagen Inc., Madison, WI). Additional primer sets were used to amplify VHH and insert a second VHH sequence with a GS linker between them to make a bivalent VHH construct. The monovalent and bivalent VHH constructs were transformed in to BL21 DE3 competent cells (New England Biolabs, Ipswich, MA). Two methods of expression and purification were utilized depending on the solubility of the VHH protein.
  • Osmotic shock For the VHH that were soluble (all VHH derived from Llama No: 2, indicated by R##), an osmotic shock protocol was utilized to purify protein from the periplasmic space, as described by Graef et al. (BMC Biotechnol 1 1, 86 (201 1)). Briefly, an ON 30 mL mid-scale culture was diluted in 450 mL Terrific Broth and grown at 25 °C for 3 hours. Cells were induced at 1 mM IPTG (Lab Scientific, Inc, Highlands, NJ) and grown for an additional 3 hours at 25 °C. After centrifugation, the cell pellet was lysed in Tris-sucrose buffer with lysozyme.
  • VHH eluted VHH were dialyzed against PBS with 1 mM DTT with at least 4 buffer changes. VHH were concentrated with Amicon Ultra- 15 Centrifugal Filter Unit (EMD Millipore, Billerica, MA), centrifuged at 16,000 x g for 10 minutes to remove precipitated protein, and protein concentration was measured by Bradford assay (BioRad, Hercules, CA).
  • EMD Millipore Billerica, MA
  • VHH sequences were amplified using primers to introduce the appropriate restriction sites for cloning into the pLEX plasmid, as well as to introduce an N-terminal His tag and C-terminal myc tag.
  • mice are injected subcutaneously in the hindquarters with 2.5 mg of Depo Provera (UpJohn Co. 400 mg/mL) seven days before the planned viral challenge.
  • the VHH candidate and the viral inoculum of 10 ID 50 are mixed in a total volume of 20 ⁇ ⁇ and promptly delivered to the vagina with a fire-polished Wiretrol pipet
  • VHHExoA [0148] Expression, Purification, Refolding of VHHExoA. [0149] A VHH that binds to gD2 of HSV-2 (called R33) was identified through the methods described above. P10, a VHH that does not bind to gD2 was also identified (SEQ ID NO: xx). VHH sequences were amplified using primers that introduced EcoRI and Xhol restriction sites for cloning in to pET-47b (Novagen Inc., Madison, WI).
  • VHHExoA protein was eluted (8 M urea, 250 mM imidazole, 50 mM NaH 2 P04, 500 mM NaCl, 300 mM DTT) diluted 1 : 100 in refolding buffer (100 mM Tris, 500 mM L-arginine, 8 mM oxidized glutathione, 2 mM EDTA), and incubated at 10 °C overnight.
  • the refolded VHHExoA was concentrated with an Amicon Ultra- 15 Centrifugal Filter Unit (EMD Millipore, Billerica, MA) and buffer exchange was performed by repeatedly bringing up the volume of the concentrated protein with PBS. The final volume of the protein was brought to ⁇ lmL, aliquoted, and frozen at -80 °C until use. Protein concentration was determined using a Bradford assay (BioRad, Hercules, CA).
  • Toxicity Assay Toxicity Assay (MTS Assay).
  • the CellTiter 96® AQueous One Solution Cell Proliferation Assay was used to determine toxicity of VHHExoA on cell lines, and the assay was carried out using the protocol recommended by the manufacturer (Promega, Madison, WI).
  • Z4/6 cells, expressing gD2 at the cell surface, and the parental L cell line were plated in 96-well trays at 3 x 10 5 cells/well overnight. The following day, dilutions of the VHHExoA proteins were added to wells and incubated overnight. About 16 hours after the addition of protein, 20 of the CellTiter 96® AQueous One Solution reagent was added to each well and incubated 4 hours at 37 °C.
  • ELISA Binding of VHHExoA to gD2. An ELISA was performed to determine if the purified VHHExoA was capable of binding to gD2. NUNC ELISA plates were coated with dilutions of VHHExoA (0.25 ⁇ g/well), and after a blocking step, dilutions of purified gD2 were added to wells in duplicate.
  • Vero cells were plated in 12-well trays at 4 x 10 6 cells/tray and after 24 hours were infected with HSV-2 G (ATCC, Manassas, VA) at 500 pfu/well. Following the 1 hour adsorption time, dilutions of the VHHExoA proteins were added to wells in duplicate and complete media (DMEM, CellGro, Manassas, VA) was added to bring volume up to 700 ⁇ ⁇ per well. About 16 hours later, supernatant was removed and cells were trypsinized briefly with 250 ⁇ ⁇ trypsin/EDTA (CellGro, Manassas, VA) before adding an equal volume of complete media.
  • HSV-2 G ATCC, Manassas, VA
  • mice are injected subcutaneously in the highquarters with 2.5 mg of Depo Provera (Up John Co. 400 mg/ml) one week before the planned viral challenge.
  • 10 ⁇ ⁇ of the virus inoculum (10 ID 50 ) is combined with ⁇ ⁇ of the VHHExoA (20 ⁇ , therefore final concentration is 10 ⁇ ) and 20 ⁇ ⁇ is promptly delivered to the vagina with a fire-polished Wiretrol pipet (Drummond Co., Broomall, PA).
  • mice Six, 24, and 48 hours post challenge and, mice are vaginally treated with a 10 ⁇ ⁇ of 20 ⁇ dose of VHHExoA.
  • the vagina On Day 10 the vagina is lavaged using 20 ⁇ of Bartel's Tissue Culture Refeeding Media; the fluid is delivered vaginally and withdrawn 10 to 20 times to collect HSV shed into the vagina.
  • the lavage fluid is centrifuged at 6500 rpm for 5 minutes to remove mucus and cells, and then placed on human newborn foreskin cells to assay for presence of virus. Cells are observed by microscope 48 hours later (Day 13) and scored yes/no for infection. All experimental procedures involving mice were approved by the Institutional Animal Care and Use
  • VHH-phage ELISA For Llama No: 1, of the 60 VHH-phage tested, there were 48 VHH- phage that reacted to gD2 by ELISA (data not shown). Sequencing revealed that 94% of the VHH sequences were identical (PI), and that overall there were 4 unique VHH sequences ( Figure 8). A VHH-phage clone, called P10, that was amplified from Llama No: 1 's library prior to any biopanning was also tested and sequenced for use as a negative control VHH- phage that did not bind to gD2. A standardized VHH-phage ELISA with 10 9 pfu per well was performed to determine relative reactivity to gD2 among the unique VHH isolated.
  • the FR2 region is critical for VHH folding and solubility because this is the region where the light chain would normally be interacting with the heavy chain, and is typically a very hydrophobic region in full-length antibodies.
  • Camelid VHH anitbodies have evolved to accumulate amino acid changes that make the region more hydrophilic, allowing for the VHH to be soluble.
  • P10 was derived from Llama No: 1, its framework sequences looks more like Llama No: 2's, indicating that there were VHH sequences present in Llama No: 1 's original library with the correct framework regions, but biopanning did not favor selection of those sequences.
  • R33 As a fusion protein with the verotoxin B subunit, which allows for self-assembly into a pentamer.
  • the verotoxin B subunit was fused to the N- terminus of R33 (NR33) and purified ( Figure 16A).
  • Figure 16A To verify that the NR33 did self-assemble, a sample of the purified NR33 was run through a Superdex200 column and it was found that they eluted at a peak of about lOOkDa, roughly the size of the expected protein (data not shown). When corrected for valency, NR33 was able to bind to gD2 as measured by ELISA at similar levels compared to monovalent R33 ( Figure 14B).
  • CF- 1 female mice were treated with Depo-provera, and one week after 10 ID 50 HSV-2 G was mixed with PBS, P10, R33 and promptly delivered to the mouse vagina. Three days later the vagina was lavaged, and fluid was plated on foreskin cells to assay for the presence of virus. At the time of animal testing, the gD2-binding VHH candidates that were available were R33 and bvR33. P10 was used as the negative control. Equivalent amounts of R33 and P 10 were mixed with virus and introduced in to the mouse vagina to determine if the VHH had any HSV-2 in vivo neutralizing capability.
  • VHHExoA immunotoxins were purified from induced BL21 cells ( Figure 21). Based on protocols published by Buchner et al for an scFv immunotoxin, the VHHExoA (R33ExoA and P lOExoA) were refolded and the antibody function was tested by ELISA. R33ExoA was still able to bind gD2 at levels comparable to R33 alone, while P10 and P lOExoA had no gD2 binding activity ( Figure 19).

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Abstract

La présente invention concerne des immunoglobulines à chaîne lourde du type VHH ou un fragment de celles-ci ayant une affinité pour un antigène cible d'intérêt, comprenant la glycoprotéine D2 (gD2) de HSV-2 ou un antigène de celle-ci, et pour des protéines d'enveloppe de VIH-1 ou un antigène de celles-ci lié à l'exotoxine A de Pseudomonas ou des fragments fonctionnels de celle-ci. L'invention concerne en outre des formes multimères des immunoglobulines et leur utilisation dans la prévention et/ou le traitement de HSV2 et/ou VIH-1.
PCT/US2015/010045 2014-01-02 2015-01-02 Compositions antimicrobiennes comprenant des anticorps monodomaines et l'exotoxine de pseudomonas WO2015103479A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220193133A1 (en) * 2020-12-18 2022-06-23 Century Therapeutics, Inc. Chimeric antigen receptor system with adaptable receptor specificity

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012019058A1 (fr) * 2010-08-05 2012-02-09 Hera Pharmaceuticals, Inc. Expression d'anticorps ou d'un fragment d'anticorps dans lactobacillus
US20130164307A1 (en) * 2010-06-17 2013-06-27 The Johns Hopkins University Use of camelid-derived variable heavy chain variable regions (vhh) targeting human cd18 and icam-1 as a microbicide to prevent hiv-1 transmission

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2753644A1 (fr) * 2011-09-09 2014-07-16 Universiteit Utrecht Holding B.V. Vhh à neutralisation large dirigés contre le vih-1

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130164307A1 (en) * 2010-06-17 2013-06-27 The Johns Hopkins University Use of camelid-derived variable heavy chain variable regions (vhh) targeting human cd18 and icam-1 as a microbicide to prevent hiv-1 transmission
WO2012019058A1 (fr) * 2010-08-05 2012-02-09 Hera Pharmaceuticals, Inc. Expression d'anticorps ou d'un fragment d'anticorps dans lactobacillus

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
GARAICOECHEA ET AL.: "Llama-derived single-chain antibody fragments directed to rotavirus VP6 protein possess broad neutralizing activity in vitro and confer protection against diarrhea in mice", JOURNAL OF VIROLOGY, vol. 82, no. 19, 2008, pages 9753 - 9764 *
GEOGHEGAN ET AL.: "Antiviral activity of a single-domain antibody immunotoxin binding to glycoprotein D of herpes simplex virus 2", ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, vol. 59, no. 1, 10 November 2014 (2014-11-10), pages 527 - 535 *
SERRUYS ET AL.: "Llama-derived single-domain intrabodies inhibit secretion of hepatitis B virions in mice", HEPATOLOGY, vol. 49, no. 1, 2009, pages 39 - 49 *
WESOLOWSKI ET AL.: "Single domain antibodies: promising experimental and therapeutic tools in infection and immunity", MEDICAL MICROBIOLOGY AND IMMUNOLOGY, vol. 198, no. 3, 2009, pages 157 - 174 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220193133A1 (en) * 2020-12-18 2022-06-23 Century Therapeutics, Inc. Chimeric antigen receptor system with adaptable receptor specificity
US11883432B2 (en) * 2020-12-18 2024-01-30 Century Therapeutics, Inc. Chimeric antigen receptor system with adaptable receptor specificity

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